HIV/HCV Coinfected's Liver Disease Can Progress Quickly: "(34%) had progression of one or more METAVIR stages between biopsies and are referred to as "progressors......Measures of obesity (BMI, P = 0.02), diabetes (P = 0.01), and hepatic steatosis (P = 0.01) at the time of the first biopsy were associated with progression of fibrosis on the subsequent liver biopsy"....genotype 1 is identified in Table 4 below as being associated with fibrosis progression\

from Jules: many of you may recall this was presented initially as an abstractat a major conference several years ago.

"In conclusion, approximately one-third of HIV/HCV coinfected patients experienced fibrosis progression of at least one METAVIR stage over a relatively short period of time, including patients with no or minimal fibrosis on first biopsy and those taking ART. Patients with persistent liver enzyme elevation, particularly of serum AST, were more likely to progress, suggesting that this simple measurement may be useful in identifying coinfected patients at greater risk for HCV disease progression. The association of obesity and its related complications with fibrosis progression underscores the potential importance of this modifiable risk factor. However, our limited ability to accurately predict progression in most patients, underscores the need for additional research to understand the basis for variable HCV disease progression in HIV-infected patients."

"The majority of coinfected patients in our prospective cohort had minimal fibrosis on initial liver biopsy. Nonetheless, over a median follow-up time of 2.5 years we observed fibrosis progression (≥1 METAVIR stage) in one-third of individual patients between the first and second liver biopsy (n = 282) and among one-third of all biopsy pairs (n = 435). While the majority of histologic change was limited to one METAVIR stage, progression of two or more stages was found in ~9% of biopsy pairs. Further,nearly 45% of patients with no evidence of hepatic fibrosis on the initial biopsy had at least stage 1 fibrosis on subsequent liver biopsy. This observed incidence of fibrosis progression over a relatively short time interval is consistent with our earlier observations and those reported in other HIV/HCV coinfected patient cohorts in which progressive disease was noted in 17% to 50% of paired histologic evaluations"

"Our finding of steatosis as a predictor of fibrosis progression is in concordance with recent investigations on this topic that have found the presence of steatosis to be strongly associated with advanced fibrosis"

"Among the 435 biopsy pairs, 149 (34%) had progression of one or more METAVIR stages between biopsies and are referred to as "progressors"(Table 3). Markers of increased hepatic inflammation measured by median AST and ALT as well as noninvasive measures of liver disease (AST-platelet ratio index [APRI] and FIB4 index) were associated with subsequent fibrosis progression.Measures of obesity (BMI, P = 0.02), diabetes (P = 0.01), and hepatic steatosis (P = 0.01) at the time of the first biopsy were associated with progression of fibrosis on the subsequent liver biopsy. Compared to nonobese patients, those with BMI >30 at the time of the initial biopsy had higher AST levels, higher prevalence of steatosis, higher histological activity indexes, and were more likely to be male "

"we did not detect an association of ART, HIV RNA suppression, or CD4 cell count with fibrosis progression. In contrast, we recently observed that the receipt of ART was independently associated with a 66% reduction in the risk of HCV-related clinical outcomes including end-stage liver disease, hepatocellular carcinoma, or liver-related death.[25] Taken together, these findings suggest that while the treatment or prevention of HIV disease may reduce liver inflammation and clinical outcomes, ART alone is not sufficient to prevent fibrosis progression in coinfected patients."

"Similar to baseline correlates, measures of hepatic inflammation were significantly different between progressors and nonprogressors between biopsies. The median AST and ALT levels between biopsies were significantly higher among biopsy pairs with fibrosis progression compared to those without fibrosis progression (P = 0.0004 for ALT and P < 0.0001 for AST). In addition, between biopsies, pairs with fibrosis progression had a significantly greater proportion of ALT and AST values >2.5 times the upper limit of normal compared with those without progression (P = 0.0002 for ALT and P < 0.0001 for AST). Time between biopsies was not associated with progression. After adjustment for baseline and between biopsy factors in multivariate analysis, the proportion of AST level >100 IU/mL between biopsies was independently associated with subsequent fibrosis progression for all biopsy pairs and for biopsy pairs restricted to those with minimal fibrosis (stage 0 or 1) (Table 4)"

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection is associated with progressive liver disease. However, the rate of progression is variable and the ability to differentiate patients with stable versus progressive HCV disease is limited. The objective of this study was to assess the incidence of and risk factors for fibrosis progression in a prospective cohort of coinfected patients. Overall, 435 liver biopsy pairs from 282 patients without cirrhosis were analyzed. Biopsies were scored according to the METAVIR system by a single pathologist blind to biopsy sequence. Fibrosis progression was defined as an increase of at least one METAVIR fibrosis stage between paired biopsies. The majority of patients were African American (84.8%), male (67.7%), and infected with HCV genotype 1 (93.4%). On initial biopsy, no or minimal fibrosis was identified in 243 patients (86%). The median interval between biopsies was 2.5 years. Fibrosis progression was observed in 97 of 282 (34%) patients and 149 of 435 (34%) biopsy pairs. After adjustment, greater body mass index (adjusted odds ratio [aOR]: 1.04 per 1 unit increase), diabetes (aOR: 1.56), and hepatic steatosis (aOR: 1.78) at the time of initial biopsy were marginally associated with subsequent fibrosis progression. Between biopsies, elevated serum aspartate and alanine aminotransferase (AST, ALT) (aOR AST: 3.34, ALT: 2.18 for >25% values >100 U/L versus <25% values >100 U/L) were strongly associated with fibrosis progression. Conclusion: Fibrosis progression is common among HIV/HCV coinfected patients; these data suggest that progression can be rapid. Persistent elevations in serum transaminase levels may serve as important noninvasive markers to identify subsets of patients who are more likely to progress and thus warrant closer monitoring and consideration of HCV treatment.

Due to shared modes of transmission, 15%-30% of individuals with human immunodeficiency virus (HIV) infection are coinfected with hepatitis C virus (HCV).[1, 2] In the era of antiretroviral therapy (ART), chronic HCV infection leads to progressive liver disease, resulting in end-stage liver disease, hepatocellular carcinoma, and death in some, but not all coinfected patients.[3-5] While the variable progression of HCV disease is well recognized, the rate and risk factors for progressive liver disease in HIV/HCV coinfected patients are incompletely understood. Several studies conducted shortly after the availability of highly active ART suggest that effective treatment of HIV may be associated with decreased risk of liver disease progression.[6-10] However, the contributions of other potentially modifiable (e.g., obesity) and unmodifiable (e.g., age) factors to the worsening of hepatic fibrosis have not been determined. Greater understanding of such factors may have important implications for the clinical management of HIV/HCV coinfected patients. For example, current HCV treatment guidelines for HIV-infected patients recommend treatment of those patients at the greatest risk for developing liver disease.

Some, but not all, expert guidelines recommend HCV treatment for HIV-infected patients independent of biopsy stage based on an assumption of rapidly progressive disease in this population.[11-17] The identification of factors associated with progression may help to refine clinical decision-making as well as identify potentially modifiable exposures. Accordingly, the objective of this study was to determine the incidence of and risk factors for fibrosis progression in a prospective cohort of coinfected adults who underwent serial liver biopsy with the aim of identifying coinfected patients with no or minimal fibrosis who are at risk for progressive liver disease over a relatively short period of time.

Patients and Methods

Study Population

This prospective cohort study evaluated 289 HIV/HCV coinfected adults who received medical care in an urban HIV clinic in Baltimore, Maryland, from July 1993 until December 2008. Treatment for HIV and/or HCV was provided by healthcare providers according to published practice guidelines.[18, 19]Individuals with at least two liver biopsies as part of their medical care were included in the study. A total of 282 patients had an initial noncirrhotic biopsy and were assessed. Of these individuals, 124 had more than two liver biopsies including 97 patients with three biopsies, 25 with four biopsies, and two with five biopsies. In total, these 282 patients contributed 435 liver biopsy pairs to the analysis. For all patients, demographic, clinical, and laboratory data were abstracted from patient charts and a laboratory database by trained personnel. Data on injection drug use and alcohol abuse were ascertained based on physician diagnosis, chart review, and self-reports.

A transcutaneous liver biopsy was performed using an 18G needle. Liver tissue was fixed in 10% formalin, and paraffin-embedded sections were stained with hematoxylin-eosin and trichome stains. Biopsies were scored according to the METAVIR and the modified histological activity index scoring system by a single pathologist (M.T.) blind to biopsy sequence. The scale to classify fibrosis was as follows: F0 = no fibrosis; F1 = portal fibrosis without septa; F2 = portal fibrosis with few septa; F3 = numerous septa without cirrhosis; F4 = cirrhosis. Steatosis was scored based on the percentage of hepatocytes affected according to a 5-point scale as follows: Grade 0: none; 1: <5% fat; 2: 5%-<30% fat; 3: 30%-60% fat; 4: >60% fat.[20] All biopsies were deemed adequate for inclusion based on expert opinion by the hepatopathologist (M.T.) including assessment of size and number of portal tracts. The median length of the first biopsy of a pair was 12.0 mm (interquartile range [IQR] 10.0, 14.0 mm), while the median length of the second biopsy of a pair was 13.0 mm (IQR 10.0, 15.0 mm). The median number of portal tracts for the first biopsy of a pair was 10 (IQR 8, 13), and for the second biopsy of a pair was 11 (IQR 9, 14).

Statistical Analysis

Significant fibrosis progression was defined as an increase of at least one METAVIR stage between the biopsies. The proportion of patients who had fibrosis progression was equivalent when analyzed using the Ishak scoring system (Supporting Table 1). Subsequently, the remaining analysis was done using the METAVIR scoring system alone. To characterize changes over time, we used 435 biopsy pairs contributed by 282 individuals such that the unit of analysis was the biopsy pair and not the individual (e.g., for a patient with three biopsies, biopsy 1 -> 2 was analyzed as one pair and biopsy 2 -> 3 as a second pair).

Univariate and multivariate logistic regression with generalized estimating equation were used to assess determinants of fibrosis progression in order to account for the correlation of biopsy pairs from within the same individuals. Variables associated with fibrosis progression in univariate analysis with P < 0.15 were considered for multivariate models. Time between biopsies was included as a covariate in the models as a categorical variable (<2, 2-2.9, 3-3.9, 4+ years). Further variables that had been previously identified as predictors of progression (gender, race, and age) were forced into models regardless of statistical significance. A series of models were built, first including fixed covariates and covariates at first biopsy and then including covariates between serial biopsies. Due to colinearity, separate models were built for laboratory values measured between serial biopsies.

Predictors of interest included fixed characteristics, characteristics at the time of the first biopsy, and characteristics between the serial biopsies. Fixed characteristics included demographics, HCV genotype, and history of alcohol or injection drug use. Characteristics at the time of the first biopsy (within six months) included diabetes, body mass index (BMI), CD4 count, HIV-RNA level, ever and cumulative ART exposure up to the first biopsy, HCV-RNA level, HCV treatment before initial biopsy, cumulative HCV treatment, duration of HCV infection (estimated by age at first injection), median AST and ALT levels, hepatic steatosis, histological necroinflammatory activity, and METAVIR score. BMI was categorized according to the standard classification system as follows: normal 18.5-24.99, overweight 25-29.99, obese ≥30. The presence of diabetes was determined by clinical diagnosis.

Predictors between biopsies included any ART use between biopsies, cumulative ART use between biopsies, change in CD4 cell count and HIV viral load, HCV treatment, change in AST and ALT levels, and change in BMI. HIV viral load and CD4 cell count between biopsies were analyzed as the proportion of CD4 cell counts that were <200 cells/μL and the proportion of HIV RNA measurements that were undetectable (<400 copies/mL). ALT and AST were examined as the cumulative proportion of ALT and AST levels more than 2.5 times the upper limit of normal reference range (AST 37 U/L; ALT 40 U/L). BMI change was defined as greater than a one unit increase or decrease in BMI.

Analyses were performed using SAS v. 9.1 software (SAS Institute, Cary, NC). Approval This study was approved by the Johns Hopkins Medicine Institutional Review Boards and written informed consent was obtained for all participants.

Results

Study Population

The demographic and clinical characteristics of the study population at initial biopsy are shown in Table 1. The median age was 44.5 years (IQR 40.5, 48.7). The majority of individuals were African American (84.8%), male (67.7%), and infected with HCV genotype 1 (93.4%). A history of injection drug use (76.6%) and alcohol abuse (48%) were frequently reported. The median BMI was 25.4 (IQR 22.5, 29.2). At the time of first biopsy most patients were receiving ART (69.2%) and had been for a median duration of 1.9 years (IQR 0, 4.3). Only 28% of patients had never received ART. The median CD4 cell count was 386 cells/μL and 15.9% had CD4 cell counts <200 cells/μL. The majority of patients (55.9%) had an HIV RNA level below the limit of detection. The median ALT and AST were 47 U/L (IQR 31, 75) and 46 U/L (IQR 33, 71), respectively. AST levels exceeding 100 U/L were observed in 14.7% (40 of 272) of patients at the time of the first biopsy and were associated with clinical history of alcohol abuse and the absence of ART (Supporting Table 2). The median HCV-RNA level was 700,000 IU/mL (IQR 500,000-1,530,000). Most patients (279 of 282, 99%) had not received HCV treatment before initial biopsy.

The median interval between biopsies was 2.5 years (IQR 2-3.2 years). Fibrosis progression was observed in 97 of 282 (34%) patients between their first and second liver biopsy. Among the 435 biopsy pairs, fibrosis progression was observed in 149 (34%), with 39 biopsy pairs (8.9%) demonstrating an increase of two or more METAVIR fibrosis stages. Notably, fibrosis progression was detected in 45% of 179 pairs in which the initial biopsy of the pair revealed no fibrosis (METAVIR stage 0; Table 2). While the majority of those with progression had stage 1 fibrosis on the second biopsy, 14 biopsy pairs (7.8%) had progression of two or more METAVIR fibrosis stages.

Correlates of Progression at Baseline

Among the 435 biopsy pairs, 149 (34%) had progression of one or more METAVIR stages between biopsies and are referred to as "progressors" (Table 3). Markers of increased hepatic inflammation measured by median AST and ALT as well as noninvasive measures of liver disease (AST-platelet ratio index [APRI] and FIB4 index) were associated with subsequent fibrosis progression. Measures of obesity (BMI, P = 0.02), diabetes (P = 0.01), and hepatic steatosis (P = 0.01) at the time of the first biopsy were associated with progression of fibrosis on the subsequent liver biopsy. Compared to nonobese patients, those with BMI >30 at the time of the initial biopsy had higher AST levels, higher prevalence of steatosis, higher histological activity indexes, and were more likely to be male (Supporting Table 3). The modified histological activity index was not related to fibrosis progression. Measures of biopsy quality including the length of the specimen were not associated with fibrosis progression. The median length was 12 mm among both nonprogressors (IQR 10, 14) and progressors (IQR 9, 14) with a Wilcoxon rank sum test P value of 0.13. Similarly, measures of HIV disease (CD4 cell count, HIV-RNA, ART exposure) at the time of initial biopsy were not significantly different among progressors and nonprogressors. After adjustment for baseline factors in multivariate analysis, AST level >100 IU/mL at the time of the first liver biopsy was independently associated with subsequent fibrosis progression for all biopsy pairs (adjusted odds ratio [aOR] 2.12, 95% confidence interval [CI] 1.06-4.26) but not for biopsy pairs restricted to those with minimal fibrosis (stage 0 or 1) at first biopsy (aOR 1.54, 95% CI 0.63-3.72) (Table 4).

Among pairs METAVIR stage 0 or 1 (n = 371) on initial biopsy, we also characterized the accuracy of elevated AST level (>100 U/L) at the time of the initial liver biopsy in prediction of fibrosis progression. The sensitivity of this threshold was low (13%), with a specificity of 93%. The positive predictive value of AST >100 U/L at baseline was 53%, whereas the negative predictive value was 68%.

Correlates of Progression Between Serial Biopsies

The univariate correlations of exposures between biopsies and progression were similar to those measures at baseline (Table 3). Antiretroviral therapy and suppression of HIV replication between biopsies were not associated with fibrosis progression. The change in CD4 cell count and the proportion of measured HIV-RNA values <400 copies/mL median IQR were also not statistically different between the two groups. Treatment for HCV infection with interferon plus ribavirin was prescribed in between 90 biopsy pairs, 58 (20.5%) of the nonprogressors, and 32 (21.5%) of the progressors (P = 0.81). Of the 90 biopsy pairs, HCV treatment resulted in durable or transient viral response in 17 instances (three in sustained virologic response [SVR] and 14 with relapse). None of the treated progressors achieved SVR, whereas SVR was achieved in three out of 58 treated nonprogressors (P = 0.19).

Similar to baseline correlates, measures of hepatic inflammation were significantly different between progressors and nonprogressors between biopsies. The median AST and ALT levels between biopsies were significantly higher among biopsy pairs with fibrosis progression compared to those without fibrosis progression (P = 0.0004 for ALT and P < 0.0001 for AST). In addition, between biopsies, pairs with fibrosis progression had a significantly greater proportion of ALT and AST values >2.5 times the upper limit of normal compared with those without progression (P = 0.0002 for ALT and P < 0.0001 for AST). Time between biopsies was not associated with progression. After adjustment for baseline and between biopsy factors in multivariate analysis, the proportion of AST level >100 IU/mL between biopsies was independently associated with subsequent fibrosis progression for all biopsy pairs and for biopsy pairs restricted to those with minimal fibrosis (stage 0 or 1) (Table 4). Additional sensitivity analyses were performed to assess the impact of restricting analysis to only the first biopsy in the pair, a change of at least two METAVIR stages, biopsy length, and number of portal tracts and the results were not significantly changed (Supporting Table 4).

Discussion

Effective ART has substantially reduced the incidence of acquired immune deficiency syndrome (AIDS)-related death among HIV-infected adults; among those coinfected with HCV, liver disease has emerged as an important cause of morbidity and mortality. While HIV infection has been consistently associated with more rapid progression of hepatic fibrosis, the mechanisms underlying this association are incompletely understood. In this context, our data related to the incidence and correlates of progressive hepatic fibrosis among 282 HIV/HCV coinfected adults who underwent serial fibrosis staging resulting in 435 paired liver biopsies provide several insights into liver disease progression in HIV/HCV coinfected patients.

The majority of coinfected patients in our prospective cohort had minimal fibrosis on initial liver biopsy. Nonetheless, over a median follow-up time of 2.5 years we observed fibrosis progression (≥1 METAVIR stage) in one-third of individual patients between the first and second liver biopsy (n = 282) and among one-third of all biopsy pairs (n = 435). While the majority of histologic change was limited to one METAVIR stage, progression of two or more stages was found in ~9% of biopsy pairs. Further, nearly 45% of patients with no evidence of hepatic fibrosis on the initial biopsy had at least stage 1 fibrosis on subsequent liver biopsy. This observed incidence of fibrosis progression over a relatively short time interval is consistent with our earlier observations and those reported in other HIV/HCV coinfected patient cohorts in which progressive disease was noted in 17% to 50% of paired histologic evaluations.[3, 9, 10, 21, 22] However, the precision of these prior estimates of progression was limited by small sample size.[9, 21, 22] For example, Schiavini et al.[9]calculated a rate of fibrosis progression of 50% based on analysis of 36 paired liver biopsies. Taken together, these data indicate that progression of HCV disease can occur in HIV/HCV coinfected persons with minimal fibrosis on initial staging. Since most patients had been HCV-infected for many years prior to this first biopsy, this observation suggests that fibrosis progression may be nonlinear in this patient population and underscores the need for serial monitoring in such patients (Supporting Figure 1). Importantly, among persons with minimal disease on first biopsy, the progression was generally limited to one METAVIR stage; as such, serial monitoring allows for the detection of individuals with progressive disease prior to the onset of clinical liver disease such as hepatocellular carcinoma or end-stage liver disease.

We also identified baseline and time-varying factors associated with fibrosis progression between biopsy pairs. Interestingly, HCV genotype 1 was associated with an increased risk of progression in some models. While it is possible that this reflects biological differences in disease related to HCV diversity, our cohort was relatively homogeneous with respect to patient (largely African American) and viral (largely genotype 1) characteristics. As such, this finding requires validation in other settings. Our data confirm the relationship of chronically elevated serum liver enzyme levels, namely AST and ALT levels, and fibrosis progression. While the biologic mechanism underlying the observed relationship of AST level and disease was not directly measured, elevated serum AST levels >100 U/L in our cohort were associated with alcohol abuse and failure to be on ART and may reflect the impact of these factors on disease progression. Similar to our prior study, elevated serum AST at baseline and between histologic assessment were independently associated with progression; HIV/HCV coinfected patients for whom measured AST levels were always <100 U/L were significantly less likely to have evidence of fibrosis progression on the next liver biopsy. Thus, AST level may represent an inexpensive, routinely obtained biomarker to identify persons at greater risk of progressive disease. Interestingly, we found that higher AST levels were associated with clinical history of alcohol abuse and the lack of treatment with antiretrovirals.

Although alcohol is clearly related to HCV disease pathogenesis, accurate assessment of alcohol intake in clinical cohorts may be challenging due to underreporting by patients. The observation that patients with elevated AST levels are at greater risk of progression may represent the effect of undetected alcohol exposure; novel alcohol biomarkers such as phosphatidylethanol or carbohydrate-deficient transferrin may be useful to further assess the contribution of underreported alcohol exposure.[23, 24] Despite the observation that ART exposure was associated with a lower likelihood of having high AST levels, we did not detect an association of ART, HIV RNA suppression, or CD4 cell count with fibrosis progression. In contrast, we recently observed that the receipt of ART was independently associated with a 66% reduction in the risk of HCV-related clinical outcomes including end-stage liver disease, hepatocellular carcinoma, or liver-related death.[25] Taken together, these findings suggest that while the treatment or prevention of HIV disease may reduce liver inflammation and clinical outcomes, ART alone is not sufficient to prevent fibrosis progression in coinfected patients.

We also found that markers of metabolic derangement at initial biopsy were associated with fibrosis progression, although the statistical significance did not persist in all models after multivariate analysis. The impact of obesity and its associated complications, namely hepatic steatosis and diabetes, have appropriately become focal points of investigation in the ART era during which time the metabolic profile of HIV-infected patients has shifted.[26] Our finding of steatosis as a predictor of fibrosis progression is in concordance with recent investigations on this topic that have found the presence of steatosis to be strongly associated with advanced fibrosis.[27-30] In fact, Gaslightwala and Bin[27] found in their investigation of 154 coinfected patients that fibrosis progression rates increased in a linear fashion with the grade of hepatic steatosis. Similarly, diabetes and insulin resistance are additional complications of obesity that have been identified as independent predictors of cirrhosis.[31, 32] Prospective studies are needed to investigate strategies to modify obesity and to assess the impact on the risk of fibrosis progression in HIV/HCV coinfected patients. In the absence of such prospective data, our findings suggest that measures to facilitate weight loss should be a priority in obese coinfected patients and those with a normal BMI should strive to maintain this.

While the major strength of our study is the prospective assessment of histologic disease progression in a large sample of coinfected patients, there are several limitations to our findings. First, our cohort consists primarily of African American patients infected with HCV genotype 1; our findings may not be generalizable to more diverse patient populations including those infected with other HCV genotypes. Second, our patients who underwent serial liver biopsies were engaged in medical care and were willing to undergo multiple liver biopsies; this patient population may differ from coinfected patients who were not referred for care. Noninvasive methods of fibrosis assessment such as liver elastography may be a useful tool to overcome this potential bias. Third, few HCV/HIV coinfected patients who were successfully treated for HCV underwent serial liver biopsy; while not unexpected, this limits our ability to assess the impact of HCV eradication on disease progression. Finally, studies based on liver biopsy are subject to sampling error and misclassification. To limit misclassification, biopsies were read as pairs by a single expert hepatopathologist who was blind to biopsy sequence. The criteria for adequacy of the biopsy specimen used in our cohort may also represent a limitation since we did not apply specific criteria for adequacy based on length or number of portal tracts. However, sensitivity analysis in which such criteria were applied did not change our findings. Finally, while it is possible that some patients with apparent fibrosis progression reflect sampling error, only 33 of 256 biopsy pairs with fibrosis on the first biopsy of the pair had evidence of fibrosis regression (12.8%), suggesting that misclassification was not common.

In conclusion, approximately one-third of HIV/HCV coinfected patients experienced fibrosis progression of at least one METAVIR stage over a relatively short period of time, including patients with no or minimal fibrosis on first biopsy and those taking ART. Patients with persistent liver enzyme elevation, particularly of serum AST, were more likely to progress, suggesting that this simple measurement may be useful in identifying coinfected patients at greater risk for HCV disease progression. The association of obesity and its related complications with fibrosis progression underscores the potential importance of this modifiable risk factor. However, our limited ability to accurately predict progression in most patients, underscores the need for additional research to understand the basis for variable HCV disease progression in HIV-infected patients.

Abstract: A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

Despite agreement that portal injections of mature hepatocytes are partially effective in patients with hereditary metabolic liver diseases [1], the goal of replacing liver transplantation with this technique remains far off. Low functioning hepatocyte availability, a 50% cell loss during the procedure and difficulties in getting transplanted hepatocytes to integrate into liver plates and proliferate are the main causes of failure. The injection of stem cells rather than mature hepatocytes has given interesting results in specific rodent models [2], but there have been few trials in humans. Strategies for replacing liver transplantation have recently focused on the in vitro construction of bio-engineered livers. Decellularization-recellularization techniques have yielded liver-like transplantable organoids in small animals [3], [4]. Much remains to be done, particularly concerning the cells used for repopulation, before this complex procedure can be applied to humans.

Until recently, it was generally believed that liver organogenesis could not be reproduced in vitro, for the construction of a new liver de novo. In this study, Takabe and coworkers from Yokohama City University were able to construct a liver in a Petri dish, by culturing hepatic endoderm cells derived from human iPSCs (iPSC-HEs) with human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs). The cells were plated in two-dimensional conditions, but the human iPSC-HEs self-organized into macroscopically visible, mechanically stable, manipulable, three-dimensional cell clusters — iPSC-derived liver buds (iPSC-LBs) — four to eight days after seeding. In vitro, cells in human iPSC-LBs expressed early hepatic marker genes and were more functional than the hepatocyte-like cells generated from hiPSCs according to conventional procedures [5], [6]. The FGF and BMP pathways, which play key roles in organogenesis, were upregulated when iPSC-HEs were co-cultured with HUVECs and MSCs. The entire process in the Petri dish closely resembled the development in vivo of liver buds from the foregut in human and mouse embryos. Human iPSC-LBs were transplanted into ectopic sites in immunodeficient mice, in which they grew and divided. Using the cranial window model, the authors observed the development of a human vasculature within the iPSC-LBs. Human blood vessels within the transplant became patent by connecting to the vessels of the mouse host at the edge of the transplant within 48h of transplantation, resulting in a vascularized organoid. Two months later, the organoid had a vascular network, including sinusoids and hepatic cord organization with tight junctions. Most of the cells in the transplanted iPSC-LBs had developed into fully mature hepatocytes, with only a small percentage retaining fetal characteristics. Metabolic studies in mice with cranial windows or capsular iPSC-LB transplants, with drugs metabolized by different pathways in mice and humans, confirmed the appropriate functioning of transplanted human liver buds. Albumin production by hiPSC-LBs increased over time and was clearly stronger than that of human adult hepatocytes transplanted to the same sites. The functionality of a mesenteric transplant of hiPSC-LBs was further investigated in two models of acute liver failure in mice. In both models, hiPSC-LB transplantation significantly increased survival (Fig. 1).

Fig. 1. General scheme of the experiments.

This constitutes a major breakthrough in the production of bioengineered livers. For the first time, a tiny, rudimentary liver has been generated from a few cells in a Petri dish, and the transplantation of this organoid ultimately protected mice against acute liver failure. The use of stromal cells to coax iPS-HEs into appropriate differentiation appears to be the key to obtaining the correct three-dimensional organization, maturation of cells and vascularization. Of course, much remains to be done to improve bud quality before this technique could be applied in clinical practice. For example, the ultrastructural organization of the bud does not entirely reflect the lobular organization of a normal liver. There is also no development of an external bile tree, potentially resulting in the accumulation of bile acids, which might jeopardize the long-term functioning of the transplanted bud. It also remains unclear whether similar results could be obtained in larger animals. Above all, such buds cannot be transplanted orthotopically. The best indication might, therefore, be the treatment of hereditary metabolic liver diseases, in which the host liver can be left in place and a genetically corrected bud implanted in the mesentery [7]. Buds could also be used as a bridging technique in patients with cirrhosis on the waiting list for transplantation, but bud generation may take too long for use in patients with acute liver failure. In addition to potential clinical applications, this study provides important information about the types of cells most suitable for liver construction, which could be applied to the recellularization of scaffolds. This technique may also prove a formidable tool for pharmacological studies. It is just one of many new technologies emerging in medical sciences. A mixture of these techniques, undoubtedly including layer-by-layer 3D printing [8], will probably ultimately be required for the de novo bioengineering of livers.

Conflict of interest

The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Patients with hepatitis C (HCV) cirrhosis and thrombocytopenia represent a particularly high-risk group for future liver decompensation, death, and hepatocellular carcinoma.1 These patients are among those who most desperately require therapy and cannot afford to wait for new treatment developments. However, these are also the patients for whom current therapies pose the highest risk of complications.2 Bone marrow suppression is a well-known complication of interferon treatment, with falling blood counts during therapy often leading to dose reductions, dose interruptions, and early cessation of treatment, all of which may lower the likelihood of attaining a sustained virologic response (SVR).3 Cirrhotic patients with significant thrombocytopenia are largely excluded from clinical trials of promising new therapies and therefore must rely on existing interferon-based regimens. Strategies to enhance rates of treatment initiation and completion have the potential to maximize SVR in this difficult-to-treat population.

Eltrombopag is a new oral platelet growth factor that acts as a thrombopoietin (TPO) receptor agonist, resulting in differentiation and proliferation of megakaryocytes. It acts in an additive fashion with endogenous TPO by binding and activating the TPO receptor through an alternate binding site.4 Eltrombopag has been studied in patients with immune thrombocytopenia purpura, cirrhosis (from any cause), and in those undergoing interferon-based therapy for HCV. To clarify whether eltrombopag would enhance rates of SVR in patients with HCV and thrombocytopenia, the ENABLE 1 and ENABLE 2 trials (Eltrombopag to initiate and maintain interferon antiviral treatment to benefit subjects with HCV-related liver disease) were carried out in North America and Europe, the results of which are published together in this edition ofGastroenterology.5

Before the ENABLE study, McHutchison et al6 evaluated the use of eltrombopag in 74 patients with HCV-related cirrhosis and platelet counts between 20,000 and 70,000/μL. Patients were randomized to increasing doses of eltrombopag (30, 50, and 75 mg) for 4 weeks before initiation of interferon and then for 12 weeks during interferon-based therapy. Between 75% and 95% of treated patients achieved the primary endpoint of an increase in platelet count to 100,000/μL during the initiation phase. Higher rates were seen in those treated with higher doses of eltrombopag. Between 36% and 65% of patients treated with eltrombopag maintained a platelet count >50,000/μL and were able to complete 12 weeks of interferon therapy compared with only 6% in the placebo group. Interestingly, no thromboembolic events were seen in this small study.

The ENABLE 1 and ENABLE 2 studies were thus undertaken to assess the effect of eltrombopag on rates of SVR in patients with HCV cirrhosis undergoing interferon-based antiviral therapy. The studies differed only in the pegylated interferon used and the corresponding platelet threshold set for initiation of treatment. Both trials recruited patients with chronic HCV with platelet counts <75,000/μL. Before starting antiviral therapy, all patients received open-label eltrombopag during the study initiation phase. Eltrombopag was initiated at a dose of 25 mg/d and increased gradually to a maximum of 100 mg/d until platelet levels crossed the recommended thresholds for initiating interferon-based therapy according to the peginterferon product label. Only patients who responded to eltrombopag were eligible for randomization in a 2:1 ratio to eltrombopag maintenance treatment during antiviral therapy or placebo (ie, antiviral therapy alone). The primary endpoint of the study was the effect of eltrombopag on the attainment of SVR. Adverse events were recorded as safety endpoints.

The patient population consisted mainly of middle-aged Caucasian men with genotype 1 infection and Child–Pugh A cirrhosis. The median platelet count at trial enrollment was 59,000/μL. Patient characteristics, including interleukin (IL)-28B status, were similar in all groups. During the initiation phase, 96%–97% of patients achieved the required platelet levels to proceed with therapy, with 86% doing so on 25 or 50 mg of eltrombopag. Adverse events were minor and included headache, nausea, and diarrhea. During the antiviral phase of the trial, a significantly higher proportion of eltrombopag-treated patients attained SVR (ENABLE 1, 23% vs 14% [P = .0064]; ENABLE 2, 19% vs 13% [P = .02]) and the treatment effect remained consistent across HCV genotypes. Patients treated with eltrombopag required fewer peginterferon dose reductions and were maintained on full-dose peginterferon for a longer amount of time. Notably, however, portal vein thrombosis (PVT) occurred more frequently in the eltrombopag-treated patients (n = 12 for eltrombopag vs n = 2 for placebo). Rates of thromboembolic complications did not correlate with platelet count or eltrombopag dose. Hepatic decompensation, specifically ascites and hepatic encephalopathy, were also more frequently seen in the eltrombopag-treated group (10% eltrombopag vs 5% placebo).

The ENABLE study was an ambitious effort to improve SVR rates in a very difficult-to-cure population. Although the study nicely confirmed that eltrombopag has potent platelet stimulatory effects, it is difficult to determine whether the improved rates of SVR seen in the trial will translate to better outcomes in general clinical practice. Study investigators were required to lower peginterferon doses according to the product labels rather than clinical judgment. Most seasoned clinicians do not strictly adhere to the thresholds in the label because clinical experience has shown that maximizing medication exposure is important and clinically significant bleeding events with moderate degrees of thrombocytopenia are very rare.7, 8 The differences in SVR were owing to greater peginterferon exposure in the eltrombopag arms. Had investigators had the freedom to adjust the peginterferon dose, it is likely that patients in the placebo arm would have received more cumulative peginterferon, which may have improved their rates of SVR. The trial design clearly favored the eltrombopag arms. The authors acknowledge this limitation in the discussion; however, it is difficult to overstate the importance of this issue in interpreting the effect of eltrombopag on treatment outcome and the overall significance of the study.

Predicting the risk of bleeding in patients with cirrhosis is complex because end-stage liver disease reduces both procoagulant and anticoagulant factors. In cirrhosis, the cause of thrombocytopenia is multifactorial. In addition to splenic sequestration resulting from portal hypertension, coating of platelets by circulating immunoglobulins may lead to increased platelet destruction by the reticuloendothelial system.9 Platelet production may also be impaired owing to reduced levels of endogenous TPO4 and HCV-related bone marrow suppression.5 However, despite the low platelet counts seen, which can fall significantly further during interferon treatment, data suggest that clinically significant bleeding is uncommon in patients with liver disease–related thrombocytopenia. This may be partially explained by effects on platelet function. In patients with cirrhosis, platelet function may be enhanced due to a decrease in production of ADAMTS13, a plasma metalloprotease that normally limits the effect of von Willebrand factor on platelets.10 Furthermore, high levels of von Willebrand factor, a common finding in patients with cirrhosis, enhance platelet adhesion to the subendothelium at sites of vascular injury.9 Other studies in patients with cirrhosis, have found that platelet counts as low as 60,000/μL are able to generate thrombin levels in the normal range.10 All of these factors enhance platelet function and may limit bleeding, even with low absolute platelet counts. Roomer et al8 recorded bleeding events in a cohort of HCV patients with and without cirrhosis treated with peginterferon and ribavirin. Although epistaxis and gingival bleeding were relatively common in patients with platelet counts of <50,000/μL, only 1 major bleeding event was recorded, which occurred at a platelet level of 65,000/μL.8 Hence, a clinically relevant platelet threshold for interferon dose reduction or cessation is not known and accurately predicting the bleeding risk of an individual patient in the office is currently very difficult. However, it is fair to say that the peginterferon product labels are relatively conservative and most clinicians would be comfortable maintaining full-dose peginterferon at platelet counts well below those recommended for dose reduction.

Even if we may be comfortable with lower platelet counts than in the product labels, there is no doubt that clinicians would sleep easier if they did not have to worry about thrombocytopenia during interferon-based therapy—but at what cost? The major concern with eltrombopag in patients with cirrhosis is the potential for an increased risk of thromboembolic complications. This was borne out in the ENABLE study with a higher number of thromboembolic events in the eltrombopag-treated group compared with those who received placebo. This phenomenon has been observed in previous studies11, 12and is biologically plausible. Interestingly, a high absolute platelet count or high dose of eltrombopag was not correlated with thromboembolic events, making it difficult to predict who is at highest risk. A post hoc analysis of a previous study11identified a platelet counts of >200,000/μL as a risk factor for thrombotic events. The most common thromboembolic event in the ENABLE study was PVT, which is a well-known complication of advanced cirrhosis. The prevalence of PVT in a large, retrospective, Italian study of 701 patients with cirrhosis was 11%13 and PVT occurs more frequently in those with more advanced disease.14 The effect of PVT on the natural history of cirrhosis is not entirely clear, with studies coming to varying conclusions. A large, retrospective study of 3295 patients awaiting liver transplantation found that the presence of PVT was an independent factor associated with death,15 whereas a prospective study of 290 patients awaiting liver transplantation did not show a significant effect of PVT on mortality.16 The effect of PVT post liver transplantation is more evident. In a recent, large, systematic review by Rodriguez-Castro et al,17 the presence of an occlusive PVT was associated with an increased 30-day and 1-year mortality post liver transplantation. This finding may be particularly relevant in the ENABLE cohort of patients, whose advanced liver disease and poor response to treatment may necessitate a future liver transplant.

Beyond PVT, there was a higher rate of hepatic decompensation among eltrombopag-treated patients. The reasons for this are not entirely clear, because it was not directly correlated with PVT or other obvious thrombotic events. Interferon-based therapy is associated with a risk of decompensation; therefore, it is conceivable that the greater cumulative exposure to interferon pushed some patients to develop hepatic complications. This study confirmed what we already knew; interferon is relatively ineffective and potentially very dangerous in patients with advanced cirrhosis.2 We were reminded of this with the introduction of first-generation protease inhibitors, for which thrombocytopenia and low albumin have been recognized as predictors of serious complications, presumably because of greater exposure to interferon in patients who might otherwise have stopped therapy earlier owing to virologic failure.18 Another intriguing possibility is that decompensation itself may be a thrombotic complication. Recently, Villa et al19 showed that low-dose enoxaparin treatment in patients with cirrhosis reduced not only PVT but also lowered the rate of hepatic decompensation and improved survival. It has been proposed that the benefits of enoxaparin may relate to prevention of microthrombi in the intrahepatic circulation. Fortunately, the rates of decompensation with eltrombopag were low, but it is conceivable that increased platelet counts may promote microthrombosis, which may be clinically relevant in a very cirrhotic liver.

Eltrombopag has also been evaluated for other treatment indications in cirrhosis. The ELEVATE study (Eltrombopag Evaluated for Its Ability to Overcome Thrombocytopenia and Enable Procedures) assessed the short-term use of eltrombopag in patients with cirrhosis and thrombocytopenia (platelet count <50,000/mm3) who required an invasive procedure.11 The primary endpoint was avoidance of platelet transfusion, and a key secondary endpoint was the occurrence of bleeding. The study demonstrated that patients treated with eltrombopag were significantly less likely to require a platelet transfusion compared with patients receiving placebo (72% vs 19%), but the rate of bleeding was not different between the 2 groups. Thromboembolic events, predominantly PVT, were more common in the treated group (odds ratio, 3.04). The ELEVATE study again confirms the potent physiologic effect of eltrombopag on platelet production but it also demonstrates that the risk of thromboembolic events is present even after short-term use. Although no clear dose or platelet level was associated with thrombosis, if one elects to use eltrombopag, it would seem prudent to use the lowest dose possible to maintain a safe platelet level.

Eltrombopag is a potentially useful tool for treating clinically relevant thrombocytopenia in patients with advanced liver disease. The ENABLE study provides further evidence that eltrombopag is effective at increasing the number of eligible patients for interferon-based therapy, as well as decreasing the number of interferon dose reductions and interruptions. However, owing to the likely difference between the very conservative study protocol and routine clinical practice for platelet count–based initiation and continuation of interferon therapy, the true effect of eltrombopag on SVR rates is uncertain. The widespread use of eltrombopag should further be tempered by the increased rates of thromboembolic events associated with its use. At present, no tools are available to accurately predict the risk of bleeding or thrombosis in an individual cirrhotic patient with thrombocytopenia or to identify in whom the benefit of eltrombopag would likely outweigh the risk. It is important to note that even with eltrombopag, the absolute rates of SVR were very low (19%–23%) and the rates of serious adverse events were high (20%) in this difficult-to-cure population. It would seem, therefore, that eltrombopag should be reserved for a carefully selected subset of patients with severe thrombocytopenia who cannot wait for new therapies and are under the care of clinicians with experience treating patients with advanced cirrhosis. If one opts to use eltrombopag, the minimum effective dose should be used. In this case, rather than a randomized, controlled trial, real-world data will ENABLE us to understand the true effect of eltrombopag on SVR, but hopefully by the time such data emerge, interferon and the need for support with eltrombopag will be a thing of the past.

A mouse embryo formed with Stimulus-Triggered Acquisition of Pluripotency (STAP) cells is seen in this undated image released by RIKEN Center for Developmental Biology on January 28, 2014. CREDIT: REUTERS/HARUKO OBOKATA/RIKEN CENTER FOR DEVELOPMENTAL BIOLOGY/HANDOUT VIA REUTERS

(Reuters) - In experiments that could open a new era in stem cell biology, scientists have found a simple way to reprogram mature animal cells back into an embryonic-like state that allows them to generate many types of tissue.

The research, described as game-changing by experts in the field, suggests human cells could in future be reprogrammed by the same technique, offering a simpler way to replace damaged cells or grow new organs for sick and injured people.

Chris Mason, chair of regenerative medicine bioprocessing at University College London, who was not involved in the work, said its approach in mice was "the most simple, lowest-cost and quickest method" to generate so-called pluripotent cells - able to develop into many different cell types - from mature cells.

"If it works in man, this could be the game changer that ultimately makes a wide range of cell therapies available using the patient's own cells as starting material - the age of personalized medicine would have finally arrived," he said.

The experiments, reported in two papers in the journal Nature on Wednesday, involved scientists from the RIKEN Center for Developmental Biology in Japan and Brigham and Women's Hospital and Harvard Medical School in the United States.

The researchers took skin and blood cells, let them multiply, then subjected them to stress "almost to the point of death", they explained, by exposing them to various events including trauma, low oxygen levels and acidic environments.

One of these "stressful" situations was simply to bathe the cells in a weak acid solution for around 30 minutes.

Within days, the scientists found that the cells had not only survived but had also recovered by naturally reverting into a state similar to that of an embryonic stem cell.

These stem cells - dubbed Stimulus-Triggered Acquisition of Pluripotency, or STAP, cells by the researchers - were then able to differentiate and mature into different types of cells and tissue, depending on the environments they were put in.

"NEW ERA"

"If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells," said Haruko Obokata, who lead the work at RIKEN.

Stem cells are the body's master cells and are able to differentiate into all other types of cells. Scientists say that by helping to regenerate tissue and potentially grow new organs, they could offer ways of tackling diseases for which there are currently only limited treatments.

Recent experimental research has seen stem cells used to create a functional human liver and to create beating heart muscle tissue.

There are two main types of stem cells: embryonic ones, harvested from embryos, and adult or iPS cells, which are taken from skin or blood and reprogrammed back into stem cells.

Because the harvesting of embryonic stem cells requires the destruction of a human embryo, the technique has been the subject of ethical concerns and protests from pro-life campaigners.

Dusko Ilic, a reader in stem cell science at Kings College London, said the Nature studies described "a major scientific discovery" and predicted their findings would open "a new era in stem cell biology".

"Whether human cells would respond in a similar way to comparable environmental cues ... remains to be shown," he said in an emailed comment. "I am sure that the group is working on this and I would not be surprised if they succeed even within this calendar year."

Robin Lovell-Badge, a stem cell expert at Britain's National Institute for Medical Research, said it would be some time before the exact nature and capabilities of the STAP cells would be fully understood by scientists - and only then would their full potential in medicine become clearer.

"But the really intriguing thing to discover will be the mechanism underlying how a low pH shock triggers reprogramming," he said. "And why does it not happen when we eat lemon or vinegar, or drink cola?"

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Hepatitis C virus (HCV) affects ∼170 million people worldwide and causes significant morbidity and mortality.[1] In high-income countries, people who inject drugs (PWID) are at greatest risk of HCV infection.[2] Until recently HCV eradication seemed unlikely, but recent advances in HCV treatment and improved understanding of the effectiveness of harm-reduction intervention effectiveness give reason for optimism. Current HCV treatments can cure ∼75% of patients and new drugs will further improve effectiveness (over 90% cure) and improve tolerability.[3] If HCV treatment can be delivered effectively to those at highest risk of onward transmission, significant reductions in future HCV cases are possible. The feasibility of disease eradication must be assessed on both scientific criteria (e.g., epidemiological susceptibility, effective and practical intervention available, and demonstrated feasibility of elimination) and political criteria (e.g., burden of disease, cost of intervention).[4] With effective, curative treatment now available, HCV meets these criteria.

Importance of Targeting PWID

To achieve eradication, public health efforts must focus on PWID, the key drivers of HCV transmission. A sustained, multipronged approach could substantially reduce HCV infection in PWID over the next 10-20 years through a focus on HCV treatment as prevention, meaning improved access to more effective and well-tolerated HCV treatment. Other major elements include increasing coverage of opiate substitution therapy (OST), needle and syringe programs (NSPs), and regular HCV screening and counseling.

PWID are highly marginalized, so effective engagement and inclusion in strategy development are critical to HCV eradication. To date, health services have been unsuccessful in channeling PWID into HCV treatment, despite evidence of willingness to be treated[5] and treatment success.[6]

HCV Treatment as Prevention

For the past decade HCV treatment has mostly involved pegylated interferon and ribavirin (PEG/RBV); however, trials of direct-acting antivirals (DAAs) show increased rates of cure, improved tolerability, and reduced duration of treatment.[3, 7, 8] The first NS3 protease inhibitors, boceprevir and telaprevir, used in combination with PEG/RBV, have already improved outcomes, with up to 75% of patients chronically infected with HCV genotype-1 being cured.[3] Emerging therapies that include next-generation NS3 protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors show great promise.[7, 8] An interferon-free 12-week DAA regimen with single daily dosing and over 90% cure is a real possibility.[3]

Highly effective and tolerable HCV therapies will make treatment as prevention feasible. This strategy will require targeting PWID, few of whom undergo HCV treatment despite increasing evidence of success.[6] The rarity of PWID undergoing treatment relates to concerns about interferon toxicity and RBV teratogenicity and unsubstantiated concerns about PWID compliance and high reinfection rates. Apart from managing adverse side effects, we know little about interventions that improve HCV treatment compliance.[9] However, increasing evidence shows that PWID are compliant when treated with PEG/RBV,[10] and compliance can only rise with improved treatment tolerability. Similarly, most evidence suggests HCV reinfection following treatment remains low.[11]

Models developed by Martin et al.[12] suggest that treating a relatively small proportion of PWID could significantly reduce HCV prevalence over 15 years, with the impact varying depending on the number treated, the background HCV prevalence, treatment efficacy, and the speed of treatment scale-up (Fig. 1). Estimated HCV prevalence halved when treatment was scaled up to 15, 40, or 76 per 1,000 PWID annually in Edinburgh (Scotland), Melbourne (Australia), and Vancouver (Canada), respectively, using DAAs. Current estimated HCV prevalence in PWID in those three jurisdictions is 25%, 50%, and 65%, respectively. Recent modeling of PWID in Vietnam also revealed treatment impact on HCV prevalence.[13]

Prevention of HCV transmission is critically important for HCV eradication. Harm-reduction strategies for PWID, notably OST and NSPs, have been partially effective in reducing HCV transmission in PWID,[14] although poor coverage has limited their impact.[15] A recent study estimated that NSPs directly averted 97,000 (∼50%) new HCV infections in Australia during 2000-2009.[14] Modeling by Vickerman et al.[16] suggests that, in a setting where HCV prevalence is 40%, scaling OST/NSP coverage up from 0% to 20%, 40%, and 60% can reduce HCV prevalence over 10 years by 13%, 24%, and 33%, respectively. However, further increments in coverage produce only marginal improvements,[16] suggesting that complementary strategies are required to substantially reduce HCV prevalence.

Treatment Access and Cost

PWID are highly marginalized and few receive HCV treatment despite increasing evidence that treatment works.[6] Effective engagement with PWID is critical to HCV eradication. Integrated multidisciplinary approaches that include clinicians, nurses and other support services, located in community-based settings or OST clinics, can increase HCV assessment and treatment.[17] Infrastructure, workforce capacity and education programs focused on PWIDs' needs are needed for timely and effective strategy implementation; currently, many primary care clinicians and health service staff know little about HCV assessment and care.[18]

Current HCV treatment is expensive and the cost of scale-up with more expensive therapies will be considerable. Visconti et al.'s[19]modeling found that treating both current and former PWID for HCV using standard PEG/RBV was cost-effective. Martin et al.'s[20] model included the broader public health benefit of reducing HCV prevalence, and showed antiviral treatment for PWID saved £521 and £2,539 per quality-adjusted life year (QALY) when baseline HCV prevalence was 20% and 40%, respectively, compared with no treatment, well below generally accepted thresholds for cost-effective interventions. Despite the cost-effectiveness of treating PWID, the actual costs of HCV treatment, particularly DAAs, will challenge governments in both developed and resource-limited settings; nonetheless, the models suggest standard HCV therapy still has considerable benefits.

Injecting Networks

Most models assume homogeneous mixing of PWID with all other PWID in the population; few consider the impact of PWIDs' social and injecting networks on HCV transmission or clearance. A recent HCV PWID network model derived from empirical data indicated that injecting networks substantially impact transmission.[21] Further modeling suggested that treating PWIDs and their immediate contacts simultaneously (as opposed to ad hoc treatment) reduces the overall number of PWID needing treatment, reducing long-term HCV prevalence and treatment costs.

HCV Vaccination

Candidate vaccines designed to prevent initial infection, reduce viral persistence in acute infection, or lead to sustained virological response (SVR) in chronic infection are in phase 2 and 3 trials.[22] However, experience with the highly effective hepatitis B vaccine suggests uptake among PWID may be low.[23] Hence, an HCV vaccine will be just one component of an HCV eradication strategy.

In conclusion, eradicating HCV in PWID is ambitious but, based on the criteria for assessing disease eradicability,[4] achievable (Table 1). Treatment costs will be substantial and recruiting sufficient PWID to treatment programs challenging. However, scale-up of HCV diagnosis and treatment with new highly efficacious and tolerable drugs, plus effective and relatively inexpensive harm reduction and prevention approaches, will considerably reduce HCV prevalence. Eradicating HCV needs a sustained, focused and multipronged approach; the time to start is now.

Author Roles: M.H. wrote the first draft of the article. All authors reviewed and edited the primary and subsequent revised versions of the article.

BACKGROUND & AIMS: Patients with cirrhosis with acute variceal bleeding (AVB) have high mortality rates (15%-20%). Previously described models are seldom used to determine prognoses of these patients, partially because they have not been validated externally and because they include subjective variables, such as bleeding during endoscopy and Child-Pugh score, which are evaluated inconsistently. We aimed to improve determination of risk for patients with AVB.

METHODS: We analyzed data collected from 178 patients with cirrhosis (Child-Pugh scores of A, B, and C: 15%, 57%, and 28%, respectively) and esophageal AVB who received standard therapy from 2007 through 2010. We tested the performance (discrimination and calibration) of previously described models, including the model for end-stage liver disease (MELD), and developed a new MELD calibration to predict the mortality of patients within 6 weeks of presentation with AVB. MELD-based predictions were validated in cohorts of patients from Canada (n = 240) and Spain (n = 221).

RESULTS: Among study subjects, the 6-week mortality rate was 16%. MELD was the best model in terms of discrimination; it was recalibrated to predict the 6-week mortality rate with logistic regression (logit, -5.312 + 0.207 • MELD; bootstrapped R(2), 0.3295). MELD values of 19 or greater predicted 20% or greater mortality, whereas MELD scores less than 11 predicted less than 5% mortality. The model performed well for patients from Canada at all risk levels. In the Spanish validation set, in which all patients were treated with banding ligation, MELD predictions were accurate up to the 20% risk threshold.

CONCLUSIONS: We developed a MELD-based model that accurately predicts mortality among patients with AVB, based on objective variables available at admission. This model could be useful to evaluate the efficacy of new therapies and stratify patients in randomized trials.

(Reuters Health) - One in three Americans with a chronic disease such as diabetes, arthritis or high blood pressure has difficulty paying for food, medications or both, according to a new study.

People who had trouble affording food were four times more likely to skip some of their medications due to cost than those who got plenty to eat, researchers found.

"This leads to an obvious tension between 'milk' or 'med,'" said Dr. Niteesh Choudhry, who worked on the study at Brigham and Women's Hospital in Boston. "If you have a fixed income, should you treat or should you eat?"

The findings are based on data collected by the 2011 National Health Interview Survey, a questionnaire that offers a snapshot of the U.S. population as a whole. Nearly 10,000 people age 20 and up filled out the survey and reported having one or more chronic illnesses like cancer, asthma, emphysema or a psychiatric illness.

Among those participants, 23 percent took their medication less often than prescribed because of the cost, 19 percent reported difficulty affording food and 11 percent said they were having trouble paying for both food and medications. In the end, about one in three had trouble affording food, medication or both.

These rates are high but are similar to figures found in previous studies, said lead author Dr. Seth Berkowitz, from Massachusetts General Hospital in Boston.

Yet the link between difficulty paying for food and for medications is a novel one.

"The idea of tradeoffs that people might make (between buying medications or food) is something we haven't seen before," said Berkowitz.

The researchers also found that patients who had difficulty paying for both food and meds were 58 percent more likely to be Hispanic or African American.

With each additional chronic illness the patients reported, their risk of having a tough time affording those items went up by 56 percent, according to the findings published in The American Journal of Medicine.

Finally, people having trouble affording medications and food were 30 percent less likely to have public, non-Medicare insurance like Medicaid, and about 60 percent less likely to participate in the Special Supplemental Nutrition Program for Women, Infants, and Children, known as WIC. This program provides supplemental food and healthcare referrals for certain women and children up to age five.

By removing some of the financial pressure from people struggling to afford food, assistance programs like WIC may also help them afford their medications, Berkowitz said.

For that reason, for people struggling to pay for either food or medications, the authors recommend looking into eligibility for food assistance programs, such as the Supplemental Nutrition Assistance Program (SNAP) and WIC, along with community support services like food banks.

When it comes to medications, there may be cheaper alternatives or assistance programs for the medication a patient is already taking.

"The most important thing people can do is talk with their doctors about it," said Berkowitz.

It's also important for people to be honest with their doctor if they are unable to afford enough food, since that may affect which medications and dosages are best.

"If you are eating very irregularly, a medication that might be perfectly safe when you are eating regularly could cause low blood sugar," or other complications, Berkowitz told Reuters Health.

If patients don't bring up the fact that they are struggling to afford medications or food, Berkowitz said, the doctor won't know to adjust medications accordingly.

He said people should "not be embarrassed or ashamed" to bring up the topic with their doctor.

SOURCE: bit.ly/1evzX7V The American Journal of Medicine, online January 21, 2014.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record

1Division of Gastroenterology and Hepatology, University of Arkansas for Medical

Sciences; 2Toronto Western Hospital Liver Center

The published epidemiological data demonstrating an inverse relationship between coffee (and potentially other caffeinated beverage) consumption and liver fibrosis and its downstream complications are weighty and rapidly accumulating. Several excellent recent reviews examine this evidence in great detail (1-3), and the overwhelming conclusion is that this inverse relationship is real – coffee drinking reduces liver fibrosis. Among the strongest studies to support this observation are the findings that, after adjustment for confounders, individuals in the highest quintile of caffeine consumption had less than one third the risk of ALT elevation of those in the lowest quintile (odds ratio (OR) 0.31, 95% CI 0.16-0.61) (4) and, perhaps more importantly, advanced liver fibrosis from chronic liver diseases of various etiologies is associated with reduced coffee and total caffeine consumption (5) with one study showing that the odds of having cirrhosis decreased with increasing daily consumption of coffee in a step-wise manner from an OR of 0.47 (95% CI 0.20-1.10) for patients consuming 1 cup of coffee per day to an OR of 0.16 (95% CI 0.05-0.50) for patients consuming 4 cups per day, compared to lifetime abstainers as the reference (OR 1.0) (6). Demonstrating the clinical significance of coffee consumption, Freedman and colleagues found that among patients with advanced fibrosis, those who consumed no coffee had a risk of hepatic decompensation or hepatocellular carcinoma (HCC) of 11.1 per 100 patient-years compared to just 6.3 per 100 patient-years in those consuming ≥ 3 cups of coffee per day, with no beneficial effect seen with tea or other sources of caffeine (7). Coffee consumption has also been shown to be associated with a lower risk of fatty liver disease (8), metabolic syndrome (9), and ultimately hepatocellular carcinoma (10). As a clinician or scientist interested in the pathogenesis of liver fibrosis, one may very well ask whether these findings are of great value.

Biological plausibility is the concept that an observed epidemiological association is “consistent with existing biological and medical knowledge” (11). This concept has long been considered a cornerstone in attempts to move epidemiological associations, even those that have been replicated on multiple occasions, to a high likelihood of causality (e.g., the now overwhelmingly accepted concept that tobacco smoking causes lung disease (12). Here we provide one of potentially several mechanisms by which coffee/caffeine consumption blocks liver fibrosis – that caffeine inhibits adenosinergic signaling in liver myofibroblasts – with strong hopes of providing biological plausibility for the observed epidemiological associations. We acknowledge fully that other potential mechanisms, such as antioxidant and anti-inflammatory properties of coffee constituents, are of possible importance; however, these concepts are not sufficiently developed at the level of observed science.

The beneficial effects of coffee and caffeine extract against liver fibrosis have been demonstrated by several studies using standard rodent models of experimental liver fibrosis induced by intoxication with dimethylnitrosamine (DMN), carbon tetrachloride (CCl4), or thioacetamide (TAA) (13-18). In almost every study, ingestion of coffee blocked toxin-induced liver fibrosis/cirrhosis. Of note, conventional filtered coffee is the form generally used in most of the published studies supporting its protective role. In contrast to the above studies, one report showed that “Turkish style” unfiltered coffee consumption not only lacks any protective effect against CCl4-induced liver fibrosis, but rather aggravates CCl4-induced hepatotoxicity with significant AST and ALT elevation (19). Of note, the mechanism(s) underlying these differences was not studied, so more definitive animal experiments are highly warranted.

One mechanism by which coffee may protect against liver fibrosis is via alterations of liver signaling or inflammation. Transforming growth factor-β (TGF-β) is a major liver regulatory cytokine secreted in large quantities in standard rodent liver fibrosis models (20). TGF-β levels are reduced by coffee and caffeine administration to rats subjected to CCl4-, DMN-, and TAA-induced liver fibrosis (13-18). One of the most significant downstream effects of TGF-β signaling is the activation of hepatic stellate cells (HSC) (21). In normal liver, HSC are vitamin A-rich, lipid-storing cells present in the space of Disse (22-24). In fibrosing liver, HSC undergo myofibroblastic differentiation and markedly upregulate secretion of extracellular matrix proteins, a process commonly known as HSC activation (24). When liver fibrosis models are performed on rodents exposed to coffee, total liver collagen contents are decreased (13-15, 18).

Activated HSC also secrete matrix metalloproteinases (MMPs), whose activity is essential to maintain the balance between tissue repair and scar formation in fibrotic livers (25). Total liver MMP secretion and activity are decreased by coffee consumption (13, 14). Expression of alpha-smooth muscle actin (α-SMA) protein is commonly used as a marker of HSC activation in the fibrotic liver (24). In the presence of coffee and caffeine, α-SMA total liver expression is diminished (13, 16, 18), potentially being indicative of reduced activation of HSCs and disease progression. Altogether, the in vivo studies reviewed here show that the anti-fibrotic properties of coffee/caffeine converge at a point in which HSC activation is diminished, providing biologic plausibility for the human studies cited above.

As noted above, coffee contains myriad chemical substances that could potentially be anti-fibrotic. A number of studies using experimental liver models have specifically addressed this question, by administration of decaffeinated coffee or caffeine solution to animals (13, 16, 19). Non-coffee caffeine was shown to protect liver against fibrosis in both TAA- and CCl4-induced liver fibrosis in rats (16, 19, 26). On the other hand, several studies demonstrate that decaffeinated coffee is also protective, but to a lower extent than caffeinated coffee in experimental animals (13, 19). Taken together, it appears that there are noteworthy holes in the animal liver fibrosis literature; there are simply not enough data to make firm conclusions about the relative importance of coffee caffeine content. At present, while it is premature to assume that the major effect of coffee is mediated by caffeine, the preponderance of evidence would suggest that this is the case.

Caffeine and other xanthines, including theophylline, have several known biological targets. These molecules have been characterized as non-selective antagonists of adenosine receptors (AR), inhibitors of phosphodiesterases, antagonists of the GABAA receptor, and stimulators of intracellular calcium release (27). While each of these effects is relevant to multiple biological processes, this section focuses on the antagonistic effects of caffeine on adenosine receptors, since this biological effect is relevant to the pathogenesis of liver fibrosis/cirrhosis.

Extracellular adenosine acts via four G-protein-coupled receptors (GPCRs), known as A1, A2a, A2b and A3 adenosine receptors to induce downstream effects (for recent review see (28, 29)). The A1AR, A2aAR and A3AR are high-affinity receptors that respond to low concentrations (>10 nM) of extracellular adenosine, while A2bAR is a low affinity receptor (>1 NM) thought to be selectively activated in pathological conditions (30). A1AR and A3AR are coupled to G proteins of the Gi/o type, leading to downregulation of cAMP-dependent signaling pathways. In contrast, A2aAR and A2bAR increase the intracellular concentration of cAMP via Gs coupling. Interestingly, A2bAR can also be coupled with Gq subunit to mobilize intracellular calcium (Ca2+).

Experimental evidence of the antagonist effects of caffeine on adenosine receptors was first reported 40 years ago in the heart (31) and in the brain (32). Caffeine is a nonspecific antagonist of all adenosine receptors. Specific synthetic agonists and antagonists derived from caffeine and other xanthine compounds have been developed for each AR and are now used as research tools in the studies of their functions, as well as potential therapeutic drugs (27). This is relevant, since specific antagonists of the A2aAR inhibit experimental liver fibrosis (26, 33). In contrast, administration of A1AR, A2bAR and A3AR specific antagonists does not significantly impact liver fibrosis progression (26).

Thus, the anti-fibrotic effect of caffeine seems to be modulated by its antagonism of the A2aAR. In addition, mice lacking A2aAR expression are protected against liver fibrosis induced by CCl4 and TAA (26). A potential role of the A1AR in liver fibrosis is more controversial, as A1AR deficient mice are also protected against CCl4-induced liver fibrosis (34), but administration of the A1AR specific antagonist DPCPX has no effect (26).

HSC are well established as primary effector cells during liver fibrosis. Interestingly, human HSC express mRNA for all four adenosine receptors ((35) and Dranoff JA unpublished data), among which A2aAR is the most studied as a regulator of HSC function. Mouse HSC express all but A3AR receptors (35). Thus, HSC represent a highly plausible cellular target mediating the anti-fibrotic effect of coffee/caffeine acting via adenosine receptor antagonism. Indeed, activation of HSC A2aAR by extracellular adenosine markedly upregulates collagen secretion (26, 35, 36). Adenosinergic signaling, via A2aAR activation, redistributes stress fibers and contractile capacity in HSCs (37), likely providing a mechanism for a “stop” signal after cell migration, as evidenced by the observation that A2aAR activation blocks the chemotaxis of HSC in response to platelet derived growth factor (PDGF) (35). Finally, A2aAR activation increased HSC TGFβ secretion (35) and decreased MMP expression (26). Since all of the mechanisms listed can be blocked by caffeine, blockade of pro-fibrotic adenosinergic signaling in HSC is a reasonable explanation for the antifibrotic effects of coffee.

According to the literature presented here, coffee consumption provides protection against liver fibrosis induced by well-established chemical models. The protective mechanism seems to be mediated primarily by the action of caffeine on HSC A2aAR. However, there are holes in the literature that will need to be closed. First, since CCl4 and other pro-fibrotic chemical agents require inflammation to induce fibrosis and cirrhosis, and multiple inflammatory cell types express adenosine receptors (38, 39), the observed effects may be mediated by changes in inflammatory cell function rather than those on HSC function. Second, the animal studies performed have taken only a cursory look at the relative importance of non-caffeine coffee constituents, in part due to methodological limitations. Lastly, animal models of fibrosis are themselves analogues of human fibrosis-to-cirrhosis progression, but they are not identical. Thus, it is very possible that animal models and studies in isolated HSC will prove useful to identify biological mechanisms, but the relevance to human health will be best tested in studies of human patients.

The progression of liver injury to fibrosis to cirrhosis is a slow but deadly process. The number of North American and European patients with chronic liver disease is increasing, primarily due to steady levels of hepatitis C infection but rapidly expanding levels of fatty liver disease (primarily non-alcoholic). Thus, identification of simple measures that can slow fibrosis and prevent cirrhosis in at-risk patients is critical. Since coffee consumption appears to have salutary effects on human health overall, coffee is an attractive lifestyle measure that patients can take.

Are we ready to “write a prescription for coffee”, as asked by Torres and Harrison in a recent commentary article? (1) Most likely, the answer is yes. Our rationale is as follows. First, there is sufficient evidence to provide biological plausibility for coffee as an anti-fibrotic. Second, coffee (for most individuals) is a pleasant addition to the diet, without profound adverse effects and possibly some other health benefits (again for most individuals). Lastly, other anti-fibrotic treatments are simply lacking; they are in the pipeline, but not yet available clinically.

However, we must face caveats as well. The human studies cited suggest that the most potent observed effects of coffee require the equivalent of four or more cups per day. We are not convinced that most individuals would easily tolerate this. Moreover, if we assume that the anti-fibrotic effects of coffee are mediated by caffeine, then should patients also be offered equivalent “doses” of tea, caffeinated soft drinks, or even caffeine pills? The latter two do not seem to be consistent with contemporary health practice, and probably for good reason. Thus, at present, we would suggest that any recommendations be limited to coffee (and for reasons cited above, limited to brewed coffee).

Hopefully, the most important effect gained by the observations reviewed here is not the use of coffee as a drug, but rather the generation of testable hypotheses as to the pathogenesis, prevention, and treatment of liver fibrosis and cirrhosis.

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